DESCRIPTION: (Verbatim from the Applicant's Abstract) The members of the crotonase (enoyl CoA hydratase) superfamily catalyze a wide range of reactions related to stabilization of a thioester enolate intermediate. The five Specific Aims focus on previously unstudied reactions, thereby providing novel insights into the structural bases for mechanistic diversity within the superfamily. Each Specific Aim involves mechanistic and structural studies: the mechanistic studies will be performed in Dr. Gerlt's laboratory at the University of Illinois (P.I.); the structural studies will be performed in Dr. Hazel Holden's laboratory at the University of Wisconsin (Co-P.I.). 1) 3R-Enoyl CoA hydratase (3R-ECH) and carnitinyl CoA racemase (CaiD) likely catalyze hydration reactions of enoyl CoA substrates to generate 3R-OH acyl CoA products; the crotonase in beta-oxidation catalyzes hydration of enoyl CoA substrates to generate 3S-OH acyl CoA products. We will study both 3R-ECH and CaiD so that we can understand the structural basis for the altered stereochemistry. 2) Methylmalonyl CoA decarboxylase (MMCD; former YgfG) catalyzes the decarboxylation of methylmalonyl. We assigned this function based on enzymological studies of three enzymes encoded by an operon and have determined its high resolution x-ray structure. We will study MMCD so that we can define how the active site promotes decarboxylation. 3) 1,4-Dihydroxynaphthoyl CoA synthase (MenB) and 2-ketocyclohexanecarboxyl CoA hydrolase (BadI) share 47 percent sequence identity and catalyze Dieckman and reverse-Dieckman condensation reactions, respectively. We will study both MenB and BadI so that we can understand the structural bases for these difficult reactions. 4) Cis,cis-Muconyl isomerase (PaaG) catalyzes a reaction in phenylacetate catabolism. We will study PaaG to determine whether cis-trans isomerization involves addition of an active site nucleophile to the substrate. 5) 3-OH Isobutyryl CoA hydrolase (HIBCH) catalyzes hydrolysis of a thioester in valine catabolism. We will study HIBCH so that we can determine whether the reaction proceeds via and enolate anion or an anionic tetrahedral intermediate.